Correlation of T follicular helper cells and plasmablasts with the development of organ involvement in patients with IgG4-related disease

Correlation of T follicular helper cells and plasmablasts with the development of organ... Abstract Objective To assess the role of an abnormal immune network in the pathology of IgG4-related disease (IgG4-RD). Methods Sixteen patients diagnosed with IgG4-RD at our institution were selected. Peripheral immunocompetent cells were immunophenotyped by multicolour flow cytometry to assess the association between clinical manifestation and pathological findings. Results Compared with healthy controls, IgG4-RD patients showed comparable proportions of Th1 and Th17 cells, but higher proportions of Treg and follicular helper T (Tfh) cells. Further, the proportions of class-switched memory B cells and plasmablasts were higher in patients. Among all phenotypes, in particular, the plasmablast proportion increased from 4.2% (controls) to 16.5% (patients). The serum IgG levels were found to be correlated with the proportions of plasmablasts and Tfh cells, but not with those of other T cell subsets. In patients with extraglandular symptoms, only plasmablasts, Tfh cells and memory Treg cells were increased. Histopathological examination revealed a marked Tfh (CD4+ Bcl6+) cell infiltration; the increase of Tfh cells in the peripheral blood thus reflected the degree of Tfh cell infiltration into the tissue. Although steroid therapy reduced plasmablast and Tfh cell proportions, the memory Treg cell proportion remained unchanged. Conclusion The association found between Tfh cells and plasmablasts, linked with biological plausibility, suggests that Tfh cells contribute to the pathogenesis of IgG4-RD. Our results also suggested that controlling the Tfh cell–plasmablast axis could be a novel therapeutic strategy for treating IgG4-RD. IgG4-related disease, follicular helper T cell, plasmablast, the Human Immunology Project Rheumatology key messages Follicular helper T cells and plasmablasts were positively correlated in peripheral blood of IgG4-related disease patients. Follicular helper T cells and plasmablasts were decreased after treatment with GCs in patients with IgG4-related disease. Memory Treg cells remained unchanged after treatment with GCs in patients with IgG4-related disease. Introduction IgG4-related disease (IgG4-RD) is characterized by hyperimmunoglobulinaemia G4, marked infiltration of IgG4-positive plasma cells into swollen or hypertrophic organs, and fibrosis [1]. Although sensitive to glucocorticoid treatment, the disease has been reported to relapse in nearly half the cases [2]. It is important to elucidate the pathology of this disease and to develop novel treatment strategies for its specific pathological features. The Comprehensive Diagnostic Criteria for IgG4-RD [3] are commonly used for the diagnosis of IgG4-RD, and are characteristically based on elevated serum IgG4 levels and pathological findings. The histopathological findings include infiltration of lymphocytes and IgG4-positive plasmacytes, along with characteristic fibrosis and sclerosis of tissues [4]. According to the diagnostic criteria, production of antibodies due to excessive immune response and fibrosis are two main pathological features of IgG4-RD. Furthermore, the involvement of human leucocyte antigens such as HLA DRB 1*04:05-DQB1*04:01 and other genetic factors such as FCRL3, CTLA4 and KCNA3 has been suggested especially in autoimmune pancreatitis [5, 6], but has not been confirmed in whole IgG4-RD. Familial occurrence has not been reported for this disease. Thus, immunophenotyping in individual cases is important for elucidating the pathology of this disease. The importance of some immune cells in IgG4-RD has been reported. Oligoclonal expansion of plasmablasts has been reported in IgG4-RD [7], and this increase is not associated with serum IgG4 levels [8]. We had previously reported that the increased plasmablasts were decreased by glucocorticoid therapy in IgG4-RD [9]. Increased transitional B cells, which are important in germinal centre reactions [10], may play an important role [11], although this is controversial [12]. T cells have also been reported to be involved in IgG4-RD. For example, the importance of CD4-positive cytotoxic T cells (CTL) [13, 14], the involvement of Th2 cells in IgG4-RD cases complicated by atopy [15, 16], and the involvement of Treg cells [17, 18] and follicular helper T (Tfh) cells including Tfh2 cells and Tfh17 cells [19–21] have also been reported. Such studies that elucidate the disease pathology provide theoretical support for novel therapeutic approaches for treating IgG4-RD, such as B cell depletion therapy [22, 23]. However, because previous studies focused on a single or several immunocompetent cells, the importance of immunocompetent cells in the immune network and their relationships among themselves are unknown. To address this issue, we designed the present study, in which we immunophenotyped T cells, B cells, dendritic cells (DCs), monocytes and natural killer (NK) cells from the peripheral blood of patients with IgG4-RD according to the Human Immunology Project (HIP) Consortium programme established by the National Institute of Allergy and Infectious Diseases [24]. We then assessed the association between immune phenotypes and the disease pathology, and their changes before and after treatment. This is the first report to describe comprehensive immunophenotyping in patients with IgG4-RD. Methods Patients IgG4-RD patients who fullfilled the classfication criteria for IgG4-RD [3] (except for one patient who was diagnosed according to IgG4-related Mikulicz’s disease [25]) were enrolled in this study between April 2013 and 2015. Biopsies were performed and the diagnoses were confirmed in all patients. We also included age- and sex-matched healthy individuals as the control group. The Human Ethics Review Committee of our university reviewed and approved this study, including the collection of peripheral blood samples from the healthy control and IgG4-RD patients. Each subject provided a signed consent form. Patients’ baseline characteristics The present study included 16 patients with IgG4-RD and 26 healthy controls (Table 1). The mean age of the subjects was 60 years, and ∼70% were men. The mean disease duration was 1.6 years, while the mean serum IgG level was 2735.5 mg/dl and the mean serum IgG4 level was 693.8 mg/dl. Biopsies were performed and the diagnoses were confirmed in all patients (supplementary Table S1, available at Rheumatology Online). Further, no statistical differences were observed in the mean age and sex ratio of patients and healthy controls. Table 1 Baseline clinical characteristics of patients Variable  IgG4-RD (n = 16)  Healthy control (n = 26)  P-value  Age  60.0 (17.5)  60.6 (14.6)  0.90  Male, n (%)  11 (68.9)  13 (50.0)  0.23  Disease duration, years  1.6 (2.6)  —    Serum IgG, mg/dl  2735.5 (268.6)  —    Serum IgG4, mg/dl  693.8 (545.9)  —    CRP, mg/dl  0.7 (1.8)  —    Variable  IgG4-RD (n = 16)  Healthy control (n = 26)  P-value  Age  60.0 (17.5)  60.6 (14.6)  0.90  Male, n (%)  11 (68.9)  13 (50.0)  0.23  Disease duration, years  1.6 (2.6)  —    Serum IgG, mg/dl  2735.5 (268.6)  —    Serum IgG4, mg/dl  693.8 (545.9)  —    CRP, mg/dl  0.7 (1.8)  —    Data are mean (s.d.) unless otherwise stated. Treatment of IgG4-RD The changes in the immune phenotypes from before and after treatment were assessed in 9 of the 16 patients who participated in the study. All nine patients received glucocorticoid at an initial dose of 0.6–0.8 mg/kg and the dosages were gradually reduced. One patient received ciclosporin and another patient received MTX. The other 7 of the 16 patients were excluded since they had been followed up without treatment or were transferred to another hospital. Clinical measurement Organ involvement was assessed in each patient. To assess the association between disease activity and these immune phenotypes, patients were divided into those with and without extraglandular symptoms (glandular means parotid gland, glandula submandibularis and salivary gland). Laboratory tests including IgG, IgG4 and CRP were also evaluated. Flow cytometric analysis Flow cytometric analysis was performed as previously described [26, 27]. Briefly, peripheral blood mononuclear cells were isolated. Peripheral blood mononuclear cells were resuspended in PBS/3% human IgG (Baxter International Inc., Vienna, Austria) in order to block Fc receptors and prevent non-specific antibody binding, and then incubated for 15 min at 4 °C in the dark. Afterwards, the cells were washed with PBS containing 1% bovine serum albumin. Background fluorescence was assessed using appropriate isotype- and fluorochrome-matched control monoclonal antibodies. After staining with the indicated antibodies (supplementary Table S2, available at Rheumatology Online), cells were analysed by multicolor flow cytometry (FACSVerse; BD Biosciences, San Jose, CA, USA), and were analysed with FlowJo software (Tree Star, Ashland, OR, USA). Gating strategy of flow cytometric analysis The phenotype of immune cell subsets was defined based on the HIP protocol of comprehensive eight-colour flow cytometric analysis proposed by National Institutes of Health (NIH)/ the Federation of Clinical Immunology Societies (FOCIS) with some modification for detecting Tfh cells [24]. Details of the gating strategy of flow cytometric analysis are described in supplementary Figs S1 and S2, available at Rheumatology Online. The clones and company names of the antibodies that were used in this study are described in supplementary Table S3, available at Rheumatology Online. Immunohistochemical staining Paraffin-embedded specimens from biopsy were studied by immunohistochemistry to define the presence of positive cells. Anti-Bcl-6 (BioLegend, San Diego, CA, USA) and anti-CD4 (Abcam, Cambridge, UK) were used to define Tfh cells. Expression of both Bcl-6 and CD4 was detected by double-staining, as previously described [28]. Briefly, paraffin-embedded sections were treated in xylene and rehydrated by a gradient of ethanol. For the antigen retrieval, the microwave method was used. Blocking with 5% goat serum (except goat anti-human primary Ab) was performed. Endogenous peroxidase activity was blocked with 3% H2O2 for 30 min. Development of the Bcl-6 Ab with 2,4-diaminobutyric acid (Dako, Santa Clara, CA, USA) was performed first; CD4 Ab was incubated, with binding of secondary AP polymer-conjugated antibody and development with VECTOR Blue (Vector Laboratories, Burlingame, CA, USA). Nuclei were counterstained with haematoxylin (Dako). NanoZoomer digital pathology (Hamamatsu, Shizuoka, Japan), which converts a histology slide into a high-resolution digital slide (190 million pixels), was utilized for analysis. The density of positive cells (per square millimetre) in whole specimen was counted with NanoZoomer Digital Pathology apparatus (Hamamatsu) viewing. To confirm the quality of double staining of CD4 (clone; BC/1F6) and Bcl-6 (clone; IG191E/A8), we performed the following examination. First, the double staining of CD4 and Bcl6 or mouse IgG was performed in human tonsil section. The result was that CD4 positive T cells were around the Bcl-6 positive cell zone (supplementary Fig. S3, available at Rheumatology Online). Next, double staining of Bcl-6 and CD20 (Abcam, clone; L26) or CD21 (Abcam, clone; EP3093) was performed in the salivary glands. CD20 and Bcl-6 showed a high concordance rate (supplementary Fig. S4A, available at Rheumatology Online). In addition, CD21 positive cells, which were stained in the germinal centre, were also stained by Bcl-6 (supplementary Fig. S4B, available at Rheumatology Online). Statistical analysis Differences between groups were compared using the Mann–Whitney U test. Correlation analysis was performed using Spearman's rank correlation coefficient. A heat map of the correlation coefficients summarized the pairwise correlative relationships (blue for negative correlation and red for positive correlation), as shown in Fig. 2. Wilcoxon’s signed-rank test was used to detect statistically significant differences between baseline data and those measured at week 52. All reported P-values were two-sided and were not adjusted for multiple testing. The level of significance was set at P < 0.05. All analyses were conducted using JMP v. 11.0 (SAS Institute Inc., Cary, NC, USA) or SPSS Statistics v. 22.0 (IBM Corp., Armonk, NY, USA). Results High proportion of plasmablasts, Tfh and Treg cells in IgG4-RD patients Figure 1 shows the overall proportions of the different immune cells by phenotype in the peripheral blood of patients. First, regarding the differentiation of Th cells, the proportion of those in the effector phase, which are considered terminally differentiated effector memory T cells, was significantly higher in patients than in healthy controls (4.6% vs 3.7%). Regarding the differentiation of CTLs, the proportion of central memory CTLs was lower in patients than in healthy controls (7.9% vs 15.3%). The proportions of Th1, Th17, Treg and Tfh cells, which are subsets of Th cells, were also assessed. The proportions of Th1 and Th17 cells in patients were similar to those in healthy controls, whereas the proportions of Treg and Tfh cells in patients were significantly higher than those in healthy controls (5.3% vs 4.0% and 1.5% vs 1.0%, respectively). The proportions of all Treg cell subtypes, activated, naive and memory Treg cells, were higher in patients than in healthy controls. Fig. 1 View largeDownload slide Differences in immunophenotypes between patients with IgG4-related diseases and sex-matched healthy control subjects P-values by Mann–Whitney U-test. The extent of changes, compared with the healthy control, is shown by colour (blue for decrease and red for increase). IgG4-RD, IgG4-related disease. Fig. 1 View largeDownload slide Differences in immunophenotypes between patients with IgG4-related diseases and sex-matched healthy control subjects P-values by Mann–Whitney U-test. The extent of changes, compared with the healthy control, is shown by colour (blue for decrease and red for increase). IgG4-RD, IgG4-related disease. Regarding the differentiation of B cells, the proportion of IgM memory B cells was lower in patients than in healthy controls (13.1% vs 20.6%), whereas those of class-switched memory B cells was higher (23.7% vs 12.5%). Among all the phenotypes of immunocompetent cells, the proportion of plasmablasts, in particular, characteristically increased to 16.5% in patients, compared with 4.2% in healthy controls (Fig. 1). Regarding DCs, although no difference was observed in the proportion of plasmacytoid DCs, the proportion of myeloid DCs was lower in patients than in healthy controls (66.9% vs 80.7%). No statistically significant differences were observed in the proportions of NK cells or monocytes. These findings revealed that both T and B cells underwent abnormal differentiation at a higher frequency in patients with IgG4-RD. Correlation among plasmablasts, Tfh and memory Treg cells Next, the correlations among these immunocompetent cells were assessed. When the correlations were clustered statistically, T and B cell populations at the similar differentiation stage were found to be clustered together (Fig. 2A). Furthermore, the proportion of plasmablasts in patients, which was markedly different from that in healthy controls, formed a statistical cluster with the proportion of Tfh cells. The correlations between the T cell subsets (Th1, Th17, Treg and Tfh cells) and the plasmablasts are shown in Fig. 2B. Among the T cell subsets, only Tfh cells were positively correlated with plasmablasts. Other immune phenotypes also showed significant correlations (supplementary Table S4, available at Rheumatology Online), such as the memory Treg cells, which were correlated with plasmablasts (supplementary Fig. S5, available at Rheumatology Online). Fig. 2 View largeDownload slide The correlation among immune phenotypes Wilcoxon’s signed-rank test for the frequencies of immune cells was perfromed for the differentiation stage classified as the naïve or memory phase or activation status and lineage of functional subsets. (A) The heat maps of the correlation coefficients summarizes the pairwise correlative relationships (blue for negative correlation and red for positive correlation). (B) The correlations between the T cell subsets (Th1, Th17, Treg and Tfh cells) and the plasmablasts. Fig. 2 View largeDownload slide The correlation among immune phenotypes Wilcoxon’s signed-rank test for the frequencies of immune cells was perfromed for the differentiation stage classified as the naïve or memory phase or activation status and lineage of functional subsets. (A) The heat maps of the correlation coefficients summarizes the pairwise correlative relationships (blue for negative correlation and red for positive correlation). (B) The correlations between the T cell subsets (Th1, Th17, Treg and Tfh cells) and the plasmablasts. Proportions of plasmablasts, Tfh and memory Treg cells correlated with disease activity To determine the association of the plasmablast–Tfh cell and plasmablast–memory Treg cell axes with clinical symptoms, the correlations between phenotypes of immune cells in peripheral blood and the clinical signs were assessed. First, when the correlations of serum IgG and IgG4 levels with immune phenotypes were assessed (supplementary Table S5, available at Rheumatology Online), the proportions of Tfh cells and plasmablasts were found to be correlated with the serum IgG levels (Fig. 3A). Likewise, central memory Th cells correlated with both serum IgG levels and IgG4 levels, while naive T helper cells and naive cytotoxic naive T cells negatively correlated with serum IgG levels and IgG4 levels. Despite a lack of significant correlations, plasmablasts tended to be associated with the serum IgG4 levels (Fig. 3A). In contrast, other Th cell subsets including the memory Treg cells did not show any correlation with serum IgG or IgG4 levels. Fig. 3 View largeDownload slide The correlation between immunce phenotypes and clinical features (A) The correlations between immnune cell subsets (plasmablasts and Tfh cells) and serum IgG level. (B) The proportion of immune subsets (plasmablasts, Tfh and memory Treg cells) in patients with and without extraglandular symptoms. Fig. 3 View largeDownload slide The correlation between immunce phenotypes and clinical features (A) The correlations between immnune cell subsets (plasmablasts and Tfh cells) and serum IgG level. (B) The proportion of immune subsets (plasmablasts, Tfh and memory Treg cells) in patients with and without extraglandular symptoms. Next, to assess the association between disease activity and these immune phenotypes, the subjects were divided into those with and without extraglandular symptoms (supplementary Table S6, available at Rheumatology Online). The proportions of plasmablasts, Tfh cells and memory Treg cells were found to be significantly increased in patients with extraglandular symptoms (Fig. 3B). Circulating Tfh cells reflected the proportion of Tfh in the biopsy site The results described in the previous sections suggested that plasmablasts and Tfh cells contributed to the production of antibodies in patients with IgG4-RD. Therefore, we examined the association between the Tfh cells in peripheral blood and those that were locally infiltrating. The Tfh cells were defined as Bcl-6-positive and CD4-positive cells in the histopathological samples (Fig. 4A); the number of Tfh cells per unit biopsy area was assessed. The proportions of Tfh cells and activated Tfh cells in the peripheral blood were positively correlated with the area occupied by Tfh cells in the samples biopsied for histopathological examination (Fig. 4B; supplementary Table S7, available at Rheumatology Online). Thus, the phenotype of the immune cells in the peripheral blood likely reflected the local immune responses. Fig. 4 View largeDownload slide The correlation between histopathological samples and peripheral blood (A) Double immunohistochemistry stainning for both Bcl-6 (brown) and CD4 (blue). Both positive cells were identified as Tfh cells. Two representative cases, which showed high proportion of Tfh (left side) or low proportion of Tfh (right side), are shown. (B) The correlations between the proportion of Tfh cells in peripheral blood and that in the biopsy site. Fig. 4 View largeDownload slide The correlation between histopathological samples and peripheral blood (A) Double immunohistochemistry stainning for both Bcl-6 (brown) and CD4 (blue). Both positive cells were identified as Tfh cells. Two representative cases, which showed high proportion of Tfh (left side) or low proportion of Tfh (right side), are shown. (B) The correlations between the proportion of Tfh cells in peripheral blood and that in the biopsy site. The proportions of plasmabasts and Tfh were decreased after treatment The changes in the immune phenotypes from before and after treatment were assessed in 9 of the 16 patients who participated in the study. Four patients who had been followed up without treatment and three patients who were transferred to another hospital were excluded. Glucocorticoid therapy reduced serum IgG4 levels (Fig. 5A) in all patients, resulting in improved clinical signs. Although no changes were observed in T cell differentiation before and after treatment (supplementary Table S8, available at Rheumatology Online), the proportions of Th17 and Tfh cells were found to be decreased (Fig. 5B). However, the proportion of memory Treg cells remained unchanged (Fig. 5B). In contrast, among the B cell subsets, IgM memory B cells were found to be increased, while plasmablasts were found to be markedly decreased (Fig. 5B). Fig. 5 View largeDownload slide Effects of treatment on the serum IgG4 level and the percentages of immune cells (A) Serum IgG4 level before and after treatment. (B) Changes in proportions of immune cell phenotypes. Values in (A) and (B) are for individual patients. The thick lines and error bar represent means and standard deviations, respectively. W, weeks. Fig. 5 View largeDownload slide Effects of treatment on the serum IgG4 level and the percentages of immune cells (A) Serum IgG4 level before and after treatment. (B) Changes in proportions of immune cell phenotypes. Values in (A) and (B) are for individual patients. The thick lines and error bar represent means and standard deviations, respectively. W, weeks. Discussion The importance of some immune cells such as plasmablasts [8] and T cells has been described in basic studies on IgG4-RD. However, because these studies focused on a single or several types of cells, the importance of the immune cells in the overall immune network and the relationships among them are unknown. The present study is the first one to show comprehensive immunophenotyping of T cells, B cells, DCs, monocytes and NK cells in the peripheral blood of patients with IgG4-RD, and the results might help resolve this issue. Comparing patients and healthy controls, the largest difference was observed in the proportion of plasmablasts. A previous study had reported that increased plasmablast levels could be an indicator for the diagnosis of IgG4-RD [8]; the present study also showed that the proportion of plasmablasts increased remarkably in the peripheral blood. Furthermore, the proportion of plasmablasts was associated with that of Tfh cells, which confirmed the importance of the plasmablast–Tfh cell axis. In addition, these cells were correlated with serum IgG levels. An important finding from the present study was that the proportion of Tfh cells in the peripheral blood reflected that of the Tfh cells that infiltrated into the actual tissue. In general, Tfh cells enter the follicles in lymph nodes, form the germinal centre with B cells, and induce the differentiation of B cells into plasmablasts. On the other hand, the present study suggested that Tfh cells also form follicular structure in tissues affected by IgG4-RD [19] and stimulate B cells in the germinal centre in lymph nodes to develop pathological features. In fact, the number of plasmablasts tended to be associated with serum IgG4 levels. Based on the assessment of phenotypes of many immune cells mentioned previously, it was assumed that Tfh cells might induce the differentiation of B cells into IgG- or IgG4-producing plasmablasts and play a central role in pathogenesis in tissues affected by IgG4-RD. Some interesting results were also obtained for Treg cells. Th2 and Treg cells have been considered to be important in the occurrence or pathogenesis of IgG4-RD [29]. In fact, enhanced expression of IL-4, IL-10 and IL-13 in the diseased tissues has been shown [18, 30]. In addition to the cases of IgG4-RD that are complicated by atopy [15], increased numbers of Th2 and Tfh2 cells in the blood were reported in patients with IgG4-RD [21]. Regarding Treg cells, although increased numbers of CD4-positive CD25high T cells in autoimmune pancreatitis [31] and Foxp3-positive Treg cells in the salivary glands in Mikulicz’s disease [17] have been reported, there is no evidence to indicate the involvement of Treg cells in the pathological features. Furthermore, because of reports on the establishment of Tfh cells in T cell subsets [32] and the reported pathogenesis based on the involvement of mainly Tfh and B cells [20, 33], the role of Treg cells was not highlighted in the pathogenesis of IgG4-RD. However, the present study showed increased proportions of Treg cells in the peripheral blood of patients with IgG4-RD. When patients were divided based on the presence of extraglandular symptoms, among all the immune phenotypes examined, only memory Treg cells were found to be increased, besides plasmablasts and Tfh cells, as mentioned previously. In addition, the proportion of memory Treg cells did not correlate at all with serum levels of IgG or IgG4, and fluctuated in a pattern different from that of the proportions of Tfh cells and plasmablasts. These interesting results were observed after treatment. When immune phenotypes before and after treatment were assessed in patients treated with glucocorticoids, the proportions of plasmablasts and Tfh cells were found to be markedly decreased and the clinical features were found to be resolved, whereas the proportion of memory Treg cells remained unchanged. These results indicated that immune phenotypes reflected the two characteristic features of IgG4-RD, that is, production of antibodies (mediated by plasmablasts and Tfh cells) and fibrosis (mediated by TGF-β). In addition, the results indicated an immune response, in which activated Treg cells tried to suppress excessive autoimmune activity in IgG4-RD. Further detailed studies are needed to examine whether the function of Treg cells is impaired in patients with IgG4-RD. One of the limitations of the present study is the limited sample size, due to the single centre study design. However, definitive diagnosis was made by biopsy in all patients, and we performed this immunophenotyping in all patients before treatment. Because diagnostic accuracy significantly affects the results in studies of such rare diseases, we preferred to conduct this study at a single institution with at least 10 rheumatologists. In addition, because this study was conducted according to the HIP proposed by the National Institute of Allergy and Infectious Disease [24], the power was too weak to detect trends in the immune phenotypes, apart from the conventional ones. In particular, we could not assess the importance of CD4-positive CTLs in the pathogenesis of IgG4-RD, which has been described recently [13]. However, the immune phenotypes that had been assessed differently at each institution were standardized in the HIP, and this facilitated the comparison of reports from different institutions and of immune phenotypes by disease. The most difficult aspect of human immunology is to determine whether the finding is a cause or consequence. Although many suggestive phenomena were observed in the present study, they were mostly correlations, beyond which anything is purely speculation. However, as with many other studies, if the therapeutic target molecules are detected, the phenomena observed in the present study may reveal themselves as either causes or consequences. In conclusion, comprehensive immunophenotyping revealed the importance of plasmablasts and Tfh cells in IgG4-RD. Furthermore, the phenotypes of immune cells in the peripheral blood were found to reflect changes in local lesions. The results suggested that Tfh cells induced the differentiation of B cells into plasmablasts and played a major role in the pathogenesis of IgG4-RD. These findings suggested not only that B cell depletion therapy could be theoretically useful, but also that searching for molecules involved in the interaction between Tfh and B cells might lead to the development of future treatment strategies specifically designed for the pathological features of IgG4-RD. Acknowledgements The author thanks all medical staff at all institutions for providing the data. The author thanks Kahoru Noda for helping with the immunohistochemistry staining and Narumi Sakaguchi for helping with flow cytometric analysis. Funding: This work was supported in part by a Grant-In-Aid for Scientific Research from the Ministry of Health, Labor and Welfare of Japan, the Ministry of Education, Culture, Sports, Science and Technology of Japan, and the University of Occupational and Environmental Health, Japan, through UOEH Grant for Advanced Research. Disclosure statement: S.K. has received speaking fees from Bristol-Myers, Pfizer and Takeda. Y.T. has received consulting fees, speaking fees and/or honoraria from Abbvie, Chugai, Daiichi-Sankyo, Bristol-Myers, Mitsubishi-Tanabe, Astellas, Takeda, Pfizer, Teijin, Asahi-kasei, YL Biologics, Sanofi, Janssen, Eli Lilly and GlaxoSmithKline and has received research grants from Mitsubishi-Tanabe, Takeda, Daiichi-Sankyo, Chugai, Bristol-Myers, MSD, Astellas, Abbvie and Eisai. All other authors have declared no conflicts of interest. Supplementary data Supplementary data are available at Rheumatology online. References 1 Kamisawa T, Zen Y, Pillai S, Stone JH. IgG4-related disease. Lancet  2015; 385: 1460– 71. http://dx.doi.org/10.1016/S0140-6736(14)60720-0 Google Scholar CrossRef Search ADS PubMed  2 Kamisawa T, Shimosegawa T, Okazaki K et al.   Standard steroid treatment for autoimmune pancreatitis. Gut  2009; 58: 1504– 7. http://dx.doi.org/10.1136/gut.2008.172908 Google Scholar CrossRef Search ADS PubMed  3 Umehara H, Okazaki K, Masaki Y et al.   Comprehensive diagnostic criteria for IgG4-related disease (IgG4-RD), 2011. Mod Rheumatol  2012; 22: 21– 30. http://dx.doi.org/10.3109/s10165-011-0571-z Google Scholar CrossRef Search ADS PubMed  4 Deshpande V, Zen Y, Chan JK et al.   Consensus statement on the pathology of IgG4-related disease. Modern Pathol  2012; 25: 1181– 92. http://dx.doi.org/10.1038/modpathol.2012.72 Google Scholar CrossRef Search ADS   5 Ota M, Katsuyama Y, Hamano H et al.   Two critical genes (HLA-DRB1 and ABCF1) in the HLA region are associated with the susceptibility to autoimmune pancreatitis. Immunogenetics  2007; 59: 45– 52. Google Scholar CrossRef Search ADS PubMed  6 Ota M, Umemura T, Kawa S. Immunogenetics of IgG4-related AIP. Curr Top Microbiol Immunol  2017; 401: 35– 44. Google Scholar PubMed  7 Mattoo H, Mahajan VS, Della-Torre E et al.   De novo oligoclonal expansions of circulating plasmablasts in active and relapsing IgG4-related disease. J Allergy Clin Immunol  2014; 134: 679– 87. http://dx.doi.org/10.1016/j.jaci.2014.03.034 Google Scholar CrossRef Search ADS PubMed  8 Wallace ZS, Mattoo H, Carruthers M et al.   Plasmablasts as a biomarker for IgG4-related disease, independent of serum IgG4 concentrations. Ann Rheum Dis  2015; 74: 190– 5. http://dx.doi.org/10.1136/annrheumdis-2014-205233 Google Scholar CrossRef Search ADS PubMed  9 Iwata S, Saito K, Hirata S, Tanaka Y. Phenotypic changes of lymphocyte in a patient with IgG4-related disease after corticosteroid therapy. Ann Rheum Dis  2012; 71: 2058– 9. http://dx.doi.org/10.1136/annrheumdis-2012-201657 Google Scholar CrossRef Search ADS PubMed  10 Achour A, Simon Q, Mohr A et al.   Human regulatory B cells control the TFH cell response. J Allergy Clin Immunol  2017; 140: 215– 22. http://dx.doi.org/10.1016/j.jaci.2016.09.042 Google Scholar CrossRef Search ADS PubMed  11 Lin W, Jin L, Chen H et al.   B cell subsets and dysfunction of regulatory B cells in IgG4-related diseases and primary Sjogren's syndrome: the similarities and differences. Arthritis Res Ther  2014; 16: R118. Google Scholar CrossRef Search ADS PubMed  12 Sumimoto K, Uchida K, Kusuda T et al.   The role of CD19+ CD24high CD38high and CD19+ CD24high CD27+ regulatory B cells in patients with type 1 autoimmune pancreatitis. Pancreatology  2014; 14: 193– 200. Google Scholar CrossRef Search ADS PubMed  13 Mattoo H, Mahajan VS, Maehara T et al.   Clonal expansion of CD4+ cytotoxic T lymphocytes in patients with IgG4-related disease. J Allergy Clin Immunol  2016; 138: 825– 38. Google Scholar CrossRef Search ADS PubMed  14 Maehara T, Mattoo H, Ohta M et al.   Lesional CD4+ IFN-γ+ cytotoxic T lymphocytes in IgG4-related dacryoadenitis and sialoadenitis. Ann Rheum Dis  2017; 76: 377– 85. Google Scholar CrossRef Search ADS PubMed  15 Mattoo H, Della-Torre E, Mahajan VS, Stone JH, Pillai S. Circulating Th2 memory cells in IgG4-related disease are restricted to a defined subset of subjects with atopy. Allergy  2014; 69: 399– 402. Google Scholar CrossRef Search ADS PubMed  16 Della Torre E, Mattoo H, Mahajan VS et al.   Prevalence of atopy, eosinophilia, and IgE elevation in IgG4-related disease. Allergy  2014; 69: 269– 72. http://dx.doi.org/10.1111/all.12320 Google Scholar CrossRef Search ADS PubMed  17 Tanaka A, Moriyama M, Nakashima H et al.   Th2 and regulatory immune reactions contribute to IgG4 production and the initiation of Mikulicz disease. Arthritis Rheum  2012; 64: 254– 63. http://dx.doi.org/10.1002/art.33320 Google Scholar CrossRef Search ADS PubMed  18 Zen Y, Fujii T, Harada K et al.   Th2 and regulatory immune reactions are increased in immunoglobin G4-related sclerosing pancreatitis and cholangitis. Hepatology  2007; 45: 1538– 46. http://dx.doi.org/10.1002/hep.21697 Google Scholar CrossRef Search ADS PubMed  19 Maehara T, Moriyama M, Nakashima H et al.   Interleukin-21 contributes to germinal centre formation and immunoglobulin G4 production in IgG4-related dacryoadenitis and sialoadenitis, so-called Mikulicz's disease. Ann Rheum Dis  2012; 71: 2011– 9. http://dx.doi.org/10.1136/annrheumdis-2012-201477 Google Scholar CrossRef Search ADS PubMed  20 Akiyama M, Yasuoka H, Yamaoka K et al.   Enhanced IgG4 production by follicular helper 2 T cells and the involvement of follicular helper 1 T cells in the pathogenesis of IgG4-related disease. Arthritis Res Ther  2016; 18: 167. http://dx.doi.org/10.1186/s13075-016-1064-4 Google Scholar CrossRef Search ADS PubMed  21 Grados A, Ebbo M, Piperoglou C et al.   T cell polarization toward TH2/TFH2 and TH17/TFH17 in patients with IgG4-related disease. Front Immunol  2017; 8: 235. Google Scholar CrossRef Search ADS PubMed  22 Khosroshahi A, Bloch DB, Deshpande V, Stone JH. Rituximab therapy leads to rapid decline of serum IgG4 levels and prompt clinical improvement in IgG4-related systemic disease. Arthritis Rheum  2010; 62: 1755– 62. Google Scholar CrossRef Search ADS PubMed  23 Carruthers MN, Topazian MD, Khosroshahi A et al.   Rituximab for IgG4-related disease: a prospective, open-label trial. Ann Rheum Dis  2015; 74: 1171– 7. http://dx.doi.org/10.1136/annrheumdis-2014-206605 Google Scholar CrossRef Search ADS PubMed  24 Maecker HT, McCoy JP, Nussenblatt R. Standardizing immunophenotyping for the Human Immunology Project. Nat Rev Immunol  2012; 12: 191– 200. Google Scholar CrossRef Search ADS PubMed  25 Masaki Y, Sugai S, Umehara H. IgG4-related diseases including Mikulicz's disease and sclerosing pancreatitis: diagnostic insights. J Rheumatol  2010; 37: 1380– 5. http://dx.doi.org/10.3899/jrheum.091153 Google Scholar CrossRef Search ADS PubMed  26 Nakayamada S, Kubo S, Yoshikawa M et al.   Differential effects of biological DMARDs on peripheral immune cell phenotypes in patients with rheumatoid arthritis. Rheumatology  2018; 57: 164– 74. Google Scholar CrossRef Search ADS PubMed  27 Kubo S, Nakayamada S, Yoshikawa M et al.   Peripheral immunophenotyping identifies three subgroups based on T cell heterogeneity in lupus patients. Arthritis Rheumatol  2017; 69: 2029– 37. Google Scholar CrossRef Search ADS PubMed  28 Zhao J, Kubo S, Nakayamada S et al.   Association of plasmacytoid dendritic cells with B cell infiltration in minor salivary glands in patients with Sjogren's syndrome. Mod Rheumatol  2016; 26: 1– 9. Google Scholar CrossRef Search ADS PubMed  29 Stone JH, Zen Y, Deshpande V. IgG4-related disease. N Engl J Med  2012; 366: 539– 51. http://dx.doi.org/10.1056/NEJMra1104650 Google Scholar CrossRef Search ADS PubMed  30 Miyake K, Moriyama M, Aizawa K et al.   Peripheral CD4+T cells showing a Th2 phenotype in a patient with Mikulicz's disease associated with lymphadenopathy and pleural effusion. Mod Rheumatol  2008; 18: 86– 90. Google Scholar CrossRef Search ADS PubMed  31 Miyoshi H, Uchida K, Taniguchi T et al.   Circulating naive and CD4+CD25high regulatory T cells in patients with autoimmune pancreatitis. Pancreas  2008; 36: 133– 40. Google Scholar CrossRef Search ADS PubMed  32 Crotty S. Follicular helper CD4 T cells (TFH). Annu Rev Immunol  2011; 29: 621– 63. http://dx.doi.org/10.1146/annurev-immunol-031210-101400 Google Scholar CrossRef Search ADS PubMed  33 Akiyama M, Suzuki K, Yamaoka K et al.   Number of circulating follicular helper 2 T cells correlates with IgG4 and interleukin-4 levels and plasmablast numbers in IgG4-related disease. Arthritis Rheumatol  2015; 67: 2476– 81. http://dx.doi.org/10.1002/art.39209 Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2017. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Rheumatology Oxford University Press

Correlation of T follicular helper cells and plasmablasts with the development of organ involvement in patients with IgG4-related disease

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Abstract

Abstract Objective To assess the role of an abnormal immune network in the pathology of IgG4-related disease (IgG4-RD). Methods Sixteen patients diagnosed with IgG4-RD at our institution were selected. Peripheral immunocompetent cells were immunophenotyped by multicolour flow cytometry to assess the association between clinical manifestation and pathological findings. Results Compared with healthy controls, IgG4-RD patients showed comparable proportions of Th1 and Th17 cells, but higher proportions of Treg and follicular helper T (Tfh) cells. Further, the proportions of class-switched memory B cells and plasmablasts were higher in patients. Among all phenotypes, in particular, the plasmablast proportion increased from 4.2% (controls) to 16.5% (patients). The serum IgG levels were found to be correlated with the proportions of plasmablasts and Tfh cells, but not with those of other T cell subsets. In patients with extraglandular symptoms, only plasmablasts, Tfh cells and memory Treg cells were increased. Histopathological examination revealed a marked Tfh (CD4+ Bcl6+) cell infiltration; the increase of Tfh cells in the peripheral blood thus reflected the degree of Tfh cell infiltration into the tissue. Although steroid therapy reduced plasmablast and Tfh cell proportions, the memory Treg cell proportion remained unchanged. Conclusion The association found between Tfh cells and plasmablasts, linked with biological plausibility, suggests that Tfh cells contribute to the pathogenesis of IgG4-RD. Our results also suggested that controlling the Tfh cell–plasmablast axis could be a novel therapeutic strategy for treating IgG4-RD. IgG4-related disease, follicular helper T cell, plasmablast, the Human Immunology Project Rheumatology key messages Follicular helper T cells and plasmablasts were positively correlated in peripheral blood of IgG4-related disease patients. Follicular helper T cells and plasmablasts were decreased after treatment with GCs in patients with IgG4-related disease. Memory Treg cells remained unchanged after treatment with GCs in patients with IgG4-related disease. Introduction IgG4-related disease (IgG4-RD) is characterized by hyperimmunoglobulinaemia G4, marked infiltration of IgG4-positive plasma cells into swollen or hypertrophic organs, and fibrosis [1]. Although sensitive to glucocorticoid treatment, the disease has been reported to relapse in nearly half the cases [2]. It is important to elucidate the pathology of this disease and to develop novel treatment strategies for its specific pathological features. The Comprehensive Diagnostic Criteria for IgG4-RD [3] are commonly used for the diagnosis of IgG4-RD, and are characteristically based on elevated serum IgG4 levels and pathological findings. The histopathological findings include infiltration of lymphocytes and IgG4-positive plasmacytes, along with characteristic fibrosis and sclerosis of tissues [4]. According to the diagnostic criteria, production of antibodies due to excessive immune response and fibrosis are two main pathological features of IgG4-RD. Furthermore, the involvement of human leucocyte antigens such as HLA DRB 1*04:05-DQB1*04:01 and other genetic factors such as FCRL3, CTLA4 and KCNA3 has been suggested especially in autoimmune pancreatitis [5, 6], but has not been confirmed in whole IgG4-RD. Familial occurrence has not been reported for this disease. Thus, immunophenotyping in individual cases is important for elucidating the pathology of this disease. The importance of some immune cells in IgG4-RD has been reported. Oligoclonal expansion of plasmablasts has been reported in IgG4-RD [7], and this increase is not associated with serum IgG4 levels [8]. We had previously reported that the increased plasmablasts were decreased by glucocorticoid therapy in IgG4-RD [9]. Increased transitional B cells, which are important in germinal centre reactions [10], may play an important role [11], although this is controversial [12]. T cells have also been reported to be involved in IgG4-RD. For example, the importance of CD4-positive cytotoxic T cells (CTL) [13, 14], the involvement of Th2 cells in IgG4-RD cases complicated by atopy [15, 16], and the involvement of Treg cells [17, 18] and follicular helper T (Tfh) cells including Tfh2 cells and Tfh17 cells [19–21] have also been reported. Such studies that elucidate the disease pathology provide theoretical support for novel therapeutic approaches for treating IgG4-RD, such as B cell depletion therapy [22, 23]. However, because previous studies focused on a single or several immunocompetent cells, the importance of immunocompetent cells in the immune network and their relationships among themselves are unknown. To address this issue, we designed the present study, in which we immunophenotyped T cells, B cells, dendritic cells (DCs), monocytes and natural killer (NK) cells from the peripheral blood of patients with IgG4-RD according to the Human Immunology Project (HIP) Consortium programme established by the National Institute of Allergy and Infectious Diseases [24]. We then assessed the association between immune phenotypes and the disease pathology, and their changes before and after treatment. This is the first report to describe comprehensive immunophenotyping in patients with IgG4-RD. Methods Patients IgG4-RD patients who fullfilled the classfication criteria for IgG4-RD [3] (except for one patient who was diagnosed according to IgG4-related Mikulicz’s disease [25]) were enrolled in this study between April 2013 and 2015. Biopsies were performed and the diagnoses were confirmed in all patients. We also included age- and sex-matched healthy individuals as the control group. The Human Ethics Review Committee of our university reviewed and approved this study, including the collection of peripheral blood samples from the healthy control and IgG4-RD patients. Each subject provided a signed consent form. Patients’ baseline characteristics The present study included 16 patients with IgG4-RD and 26 healthy controls (Table 1). The mean age of the subjects was 60 years, and ∼70% were men. The mean disease duration was 1.6 years, while the mean serum IgG level was 2735.5 mg/dl and the mean serum IgG4 level was 693.8 mg/dl. Biopsies were performed and the diagnoses were confirmed in all patients (supplementary Table S1, available at Rheumatology Online). Further, no statistical differences were observed in the mean age and sex ratio of patients and healthy controls. Table 1 Baseline clinical characteristics of patients Variable  IgG4-RD (n = 16)  Healthy control (n = 26)  P-value  Age  60.0 (17.5)  60.6 (14.6)  0.90  Male, n (%)  11 (68.9)  13 (50.0)  0.23  Disease duration, years  1.6 (2.6)  —    Serum IgG, mg/dl  2735.5 (268.6)  —    Serum IgG4, mg/dl  693.8 (545.9)  —    CRP, mg/dl  0.7 (1.8)  —    Variable  IgG4-RD (n = 16)  Healthy control (n = 26)  P-value  Age  60.0 (17.5)  60.6 (14.6)  0.90  Male, n (%)  11 (68.9)  13 (50.0)  0.23  Disease duration, years  1.6 (2.6)  —    Serum IgG, mg/dl  2735.5 (268.6)  —    Serum IgG4, mg/dl  693.8 (545.9)  —    CRP, mg/dl  0.7 (1.8)  —    Data are mean (s.d.) unless otherwise stated. Treatment of IgG4-RD The changes in the immune phenotypes from before and after treatment were assessed in 9 of the 16 patients who participated in the study. All nine patients received glucocorticoid at an initial dose of 0.6–0.8 mg/kg and the dosages were gradually reduced. One patient received ciclosporin and another patient received MTX. The other 7 of the 16 patients were excluded since they had been followed up without treatment or were transferred to another hospital. Clinical measurement Organ involvement was assessed in each patient. To assess the association between disease activity and these immune phenotypes, patients were divided into those with and without extraglandular symptoms (glandular means parotid gland, glandula submandibularis and salivary gland). Laboratory tests including IgG, IgG4 and CRP were also evaluated. Flow cytometric analysis Flow cytometric analysis was performed as previously described [26, 27]. Briefly, peripheral blood mononuclear cells were isolated. Peripheral blood mononuclear cells were resuspended in PBS/3% human IgG (Baxter International Inc., Vienna, Austria) in order to block Fc receptors and prevent non-specific antibody binding, and then incubated for 15 min at 4 °C in the dark. Afterwards, the cells were washed with PBS containing 1% bovine serum albumin. Background fluorescence was assessed using appropriate isotype- and fluorochrome-matched control monoclonal antibodies. After staining with the indicated antibodies (supplementary Table S2, available at Rheumatology Online), cells were analysed by multicolor flow cytometry (FACSVerse; BD Biosciences, San Jose, CA, USA), and were analysed with FlowJo software (Tree Star, Ashland, OR, USA). Gating strategy of flow cytometric analysis The phenotype of immune cell subsets was defined based on the HIP protocol of comprehensive eight-colour flow cytometric analysis proposed by National Institutes of Health (NIH)/ the Federation of Clinical Immunology Societies (FOCIS) with some modification for detecting Tfh cells [24]. Details of the gating strategy of flow cytometric analysis are described in supplementary Figs S1 and S2, available at Rheumatology Online. The clones and company names of the antibodies that were used in this study are described in supplementary Table S3, available at Rheumatology Online. Immunohistochemical staining Paraffin-embedded specimens from biopsy were studied by immunohistochemistry to define the presence of positive cells. Anti-Bcl-6 (BioLegend, San Diego, CA, USA) and anti-CD4 (Abcam, Cambridge, UK) were used to define Tfh cells. Expression of both Bcl-6 and CD4 was detected by double-staining, as previously described [28]. Briefly, paraffin-embedded sections were treated in xylene and rehydrated by a gradient of ethanol. For the antigen retrieval, the microwave method was used. Blocking with 5% goat serum (except goat anti-human primary Ab) was performed. Endogenous peroxidase activity was blocked with 3% H2O2 for 30 min. Development of the Bcl-6 Ab with 2,4-diaminobutyric acid (Dako, Santa Clara, CA, USA) was performed first; CD4 Ab was incubated, with binding of secondary AP polymer-conjugated antibody and development with VECTOR Blue (Vector Laboratories, Burlingame, CA, USA). Nuclei were counterstained with haematoxylin (Dako). NanoZoomer digital pathology (Hamamatsu, Shizuoka, Japan), which converts a histology slide into a high-resolution digital slide (190 million pixels), was utilized for analysis. The density of positive cells (per square millimetre) in whole specimen was counted with NanoZoomer Digital Pathology apparatus (Hamamatsu) viewing. To confirm the quality of double staining of CD4 (clone; BC/1F6) and Bcl-6 (clone; IG191E/A8), we performed the following examination. First, the double staining of CD4 and Bcl6 or mouse IgG was performed in human tonsil section. The result was that CD4 positive T cells were around the Bcl-6 positive cell zone (supplementary Fig. S3, available at Rheumatology Online). Next, double staining of Bcl-6 and CD20 (Abcam, clone; L26) or CD21 (Abcam, clone; EP3093) was performed in the salivary glands. CD20 and Bcl-6 showed a high concordance rate (supplementary Fig. S4A, available at Rheumatology Online). In addition, CD21 positive cells, which were stained in the germinal centre, were also stained by Bcl-6 (supplementary Fig. S4B, available at Rheumatology Online). Statistical analysis Differences between groups were compared using the Mann–Whitney U test. Correlation analysis was performed using Spearman's rank correlation coefficient. A heat map of the correlation coefficients summarized the pairwise correlative relationships (blue for negative correlation and red for positive correlation), as shown in Fig. 2. Wilcoxon’s signed-rank test was used to detect statistically significant differences between baseline data and those measured at week 52. All reported P-values were two-sided and were not adjusted for multiple testing. The level of significance was set at P < 0.05. All analyses were conducted using JMP v. 11.0 (SAS Institute Inc., Cary, NC, USA) or SPSS Statistics v. 22.0 (IBM Corp., Armonk, NY, USA). Results High proportion of plasmablasts, Tfh and Treg cells in IgG4-RD patients Figure 1 shows the overall proportions of the different immune cells by phenotype in the peripheral blood of patients. First, regarding the differentiation of Th cells, the proportion of those in the effector phase, which are considered terminally differentiated effector memory T cells, was significantly higher in patients than in healthy controls (4.6% vs 3.7%). Regarding the differentiation of CTLs, the proportion of central memory CTLs was lower in patients than in healthy controls (7.9% vs 15.3%). The proportions of Th1, Th17, Treg and Tfh cells, which are subsets of Th cells, were also assessed. The proportions of Th1 and Th17 cells in patients were similar to those in healthy controls, whereas the proportions of Treg and Tfh cells in patients were significantly higher than those in healthy controls (5.3% vs 4.0% and 1.5% vs 1.0%, respectively). The proportions of all Treg cell subtypes, activated, naive and memory Treg cells, were higher in patients than in healthy controls. Fig. 1 View largeDownload slide Differences in immunophenotypes between patients with IgG4-related diseases and sex-matched healthy control subjects P-values by Mann–Whitney U-test. The extent of changes, compared with the healthy control, is shown by colour (blue for decrease and red for increase). IgG4-RD, IgG4-related disease. Fig. 1 View largeDownload slide Differences in immunophenotypes between patients with IgG4-related diseases and sex-matched healthy control subjects P-values by Mann–Whitney U-test. The extent of changes, compared with the healthy control, is shown by colour (blue for decrease and red for increase). IgG4-RD, IgG4-related disease. Regarding the differentiation of B cells, the proportion of IgM memory B cells was lower in patients than in healthy controls (13.1% vs 20.6%), whereas those of class-switched memory B cells was higher (23.7% vs 12.5%). Among all the phenotypes of immunocompetent cells, the proportion of plasmablasts, in particular, characteristically increased to 16.5% in patients, compared with 4.2% in healthy controls (Fig. 1). Regarding DCs, although no difference was observed in the proportion of plasmacytoid DCs, the proportion of myeloid DCs was lower in patients than in healthy controls (66.9% vs 80.7%). No statistically significant differences were observed in the proportions of NK cells or monocytes. These findings revealed that both T and B cells underwent abnormal differentiation at a higher frequency in patients with IgG4-RD. Correlation among plasmablasts, Tfh and memory Treg cells Next, the correlations among these immunocompetent cells were assessed. When the correlations were clustered statistically, T and B cell populations at the similar differentiation stage were found to be clustered together (Fig. 2A). Furthermore, the proportion of plasmablasts in patients, which was markedly different from that in healthy controls, formed a statistical cluster with the proportion of Tfh cells. The correlations between the T cell subsets (Th1, Th17, Treg and Tfh cells) and the plasmablasts are shown in Fig. 2B. Among the T cell subsets, only Tfh cells were positively correlated with plasmablasts. Other immune phenotypes also showed significant correlations (supplementary Table S4, available at Rheumatology Online), such as the memory Treg cells, which were correlated with plasmablasts (supplementary Fig. S5, available at Rheumatology Online). Fig. 2 View largeDownload slide The correlation among immune phenotypes Wilcoxon’s signed-rank test for the frequencies of immune cells was perfromed for the differentiation stage classified as the naïve or memory phase or activation status and lineage of functional subsets. (A) The heat maps of the correlation coefficients summarizes the pairwise correlative relationships (blue for negative correlation and red for positive correlation). (B) The correlations between the T cell subsets (Th1, Th17, Treg and Tfh cells) and the plasmablasts. Fig. 2 View largeDownload slide The correlation among immune phenotypes Wilcoxon’s signed-rank test for the frequencies of immune cells was perfromed for the differentiation stage classified as the naïve or memory phase or activation status and lineage of functional subsets. (A) The heat maps of the correlation coefficients summarizes the pairwise correlative relationships (blue for negative correlation and red for positive correlation). (B) The correlations between the T cell subsets (Th1, Th17, Treg and Tfh cells) and the plasmablasts. Proportions of plasmablasts, Tfh and memory Treg cells correlated with disease activity To determine the association of the plasmablast–Tfh cell and plasmablast–memory Treg cell axes with clinical symptoms, the correlations between phenotypes of immune cells in peripheral blood and the clinical signs were assessed. First, when the correlations of serum IgG and IgG4 levels with immune phenotypes were assessed (supplementary Table S5, available at Rheumatology Online), the proportions of Tfh cells and plasmablasts were found to be correlated with the serum IgG levels (Fig. 3A). Likewise, central memory Th cells correlated with both serum IgG levels and IgG4 levels, while naive T helper cells and naive cytotoxic naive T cells negatively correlated with serum IgG levels and IgG4 levels. Despite a lack of significant correlations, plasmablasts tended to be associated with the serum IgG4 levels (Fig. 3A). In contrast, other Th cell subsets including the memory Treg cells did not show any correlation with serum IgG or IgG4 levels. Fig. 3 View largeDownload slide The correlation between immunce phenotypes and clinical features (A) The correlations between immnune cell subsets (plasmablasts and Tfh cells) and serum IgG level. (B) The proportion of immune subsets (plasmablasts, Tfh and memory Treg cells) in patients with and without extraglandular symptoms. Fig. 3 View largeDownload slide The correlation between immunce phenotypes and clinical features (A) The correlations between immnune cell subsets (plasmablasts and Tfh cells) and serum IgG level. (B) The proportion of immune subsets (plasmablasts, Tfh and memory Treg cells) in patients with and without extraglandular symptoms. Next, to assess the association between disease activity and these immune phenotypes, the subjects were divided into those with and without extraglandular symptoms (supplementary Table S6, available at Rheumatology Online). The proportions of plasmablasts, Tfh cells and memory Treg cells were found to be significantly increased in patients with extraglandular symptoms (Fig. 3B). Circulating Tfh cells reflected the proportion of Tfh in the biopsy site The results described in the previous sections suggested that plasmablasts and Tfh cells contributed to the production of antibodies in patients with IgG4-RD. Therefore, we examined the association between the Tfh cells in peripheral blood and those that were locally infiltrating. The Tfh cells were defined as Bcl-6-positive and CD4-positive cells in the histopathological samples (Fig. 4A); the number of Tfh cells per unit biopsy area was assessed. The proportions of Tfh cells and activated Tfh cells in the peripheral blood were positively correlated with the area occupied by Tfh cells in the samples biopsied for histopathological examination (Fig. 4B; supplementary Table S7, available at Rheumatology Online). Thus, the phenotype of the immune cells in the peripheral blood likely reflected the local immune responses. Fig. 4 View largeDownload slide The correlation between histopathological samples and peripheral blood (A) Double immunohistochemistry stainning for both Bcl-6 (brown) and CD4 (blue). Both positive cells were identified as Tfh cells. Two representative cases, which showed high proportion of Tfh (left side) or low proportion of Tfh (right side), are shown. (B) The correlations between the proportion of Tfh cells in peripheral blood and that in the biopsy site. Fig. 4 View largeDownload slide The correlation between histopathological samples and peripheral blood (A) Double immunohistochemistry stainning for both Bcl-6 (brown) and CD4 (blue). Both positive cells were identified as Tfh cells. Two representative cases, which showed high proportion of Tfh (left side) or low proportion of Tfh (right side), are shown. (B) The correlations between the proportion of Tfh cells in peripheral blood and that in the biopsy site. The proportions of plasmabasts and Tfh were decreased after treatment The changes in the immune phenotypes from before and after treatment were assessed in 9 of the 16 patients who participated in the study. Four patients who had been followed up without treatment and three patients who were transferred to another hospital were excluded. Glucocorticoid therapy reduced serum IgG4 levels (Fig. 5A) in all patients, resulting in improved clinical signs. Although no changes were observed in T cell differentiation before and after treatment (supplementary Table S8, available at Rheumatology Online), the proportions of Th17 and Tfh cells were found to be decreased (Fig. 5B). However, the proportion of memory Treg cells remained unchanged (Fig. 5B). In contrast, among the B cell subsets, IgM memory B cells were found to be increased, while plasmablasts were found to be markedly decreased (Fig. 5B). Fig. 5 View largeDownload slide Effects of treatment on the serum IgG4 level and the percentages of immune cells (A) Serum IgG4 level before and after treatment. (B) Changes in proportions of immune cell phenotypes. Values in (A) and (B) are for individual patients. The thick lines and error bar represent means and standard deviations, respectively. W, weeks. Fig. 5 View largeDownload slide Effects of treatment on the serum IgG4 level and the percentages of immune cells (A) Serum IgG4 level before and after treatment. (B) Changes in proportions of immune cell phenotypes. Values in (A) and (B) are for individual patients. The thick lines and error bar represent means and standard deviations, respectively. W, weeks. Discussion The importance of some immune cells such as plasmablasts [8] and T cells has been described in basic studies on IgG4-RD. However, because these studies focused on a single or several types of cells, the importance of the immune cells in the overall immune network and the relationships among them are unknown. The present study is the first one to show comprehensive immunophenotyping of T cells, B cells, DCs, monocytes and NK cells in the peripheral blood of patients with IgG4-RD, and the results might help resolve this issue. Comparing patients and healthy controls, the largest difference was observed in the proportion of plasmablasts. A previous study had reported that increased plasmablast levels could be an indicator for the diagnosis of IgG4-RD [8]; the present study also showed that the proportion of plasmablasts increased remarkably in the peripheral blood. Furthermore, the proportion of plasmablasts was associated with that of Tfh cells, which confirmed the importance of the plasmablast–Tfh cell axis. In addition, these cells were correlated with serum IgG levels. An important finding from the present study was that the proportion of Tfh cells in the peripheral blood reflected that of the Tfh cells that infiltrated into the actual tissue. In general, Tfh cells enter the follicles in lymph nodes, form the germinal centre with B cells, and induce the differentiation of B cells into plasmablasts. On the other hand, the present study suggested that Tfh cells also form follicular structure in tissues affected by IgG4-RD [19] and stimulate B cells in the germinal centre in lymph nodes to develop pathological features. In fact, the number of plasmablasts tended to be associated with serum IgG4 levels. Based on the assessment of phenotypes of many immune cells mentioned previously, it was assumed that Tfh cells might induce the differentiation of B cells into IgG- or IgG4-producing plasmablasts and play a central role in pathogenesis in tissues affected by IgG4-RD. Some interesting results were also obtained for Treg cells. Th2 and Treg cells have been considered to be important in the occurrence or pathogenesis of IgG4-RD [29]. In fact, enhanced expression of IL-4, IL-10 and IL-13 in the diseased tissues has been shown [18, 30]. In addition to the cases of IgG4-RD that are complicated by atopy [15], increased numbers of Th2 and Tfh2 cells in the blood were reported in patients with IgG4-RD [21]. Regarding Treg cells, although increased numbers of CD4-positive CD25high T cells in autoimmune pancreatitis [31] and Foxp3-positive Treg cells in the salivary glands in Mikulicz’s disease [17] have been reported, there is no evidence to indicate the involvement of Treg cells in the pathological features. Furthermore, because of reports on the establishment of Tfh cells in T cell subsets [32] and the reported pathogenesis based on the involvement of mainly Tfh and B cells [20, 33], the role of Treg cells was not highlighted in the pathogenesis of IgG4-RD. However, the present study showed increased proportions of Treg cells in the peripheral blood of patients with IgG4-RD. When patients were divided based on the presence of extraglandular symptoms, among all the immune phenotypes examined, only memory Treg cells were found to be increased, besides plasmablasts and Tfh cells, as mentioned previously. In addition, the proportion of memory Treg cells did not correlate at all with serum levels of IgG or IgG4, and fluctuated in a pattern different from that of the proportions of Tfh cells and plasmablasts. These interesting results were observed after treatment. When immune phenotypes before and after treatment were assessed in patients treated with glucocorticoids, the proportions of plasmablasts and Tfh cells were found to be markedly decreased and the clinical features were found to be resolved, whereas the proportion of memory Treg cells remained unchanged. These results indicated that immune phenotypes reflected the two characteristic features of IgG4-RD, that is, production of antibodies (mediated by plasmablasts and Tfh cells) and fibrosis (mediated by TGF-β). In addition, the results indicated an immune response, in which activated Treg cells tried to suppress excessive autoimmune activity in IgG4-RD. Further detailed studies are needed to examine whether the function of Treg cells is impaired in patients with IgG4-RD. One of the limitations of the present study is the limited sample size, due to the single centre study design. However, definitive diagnosis was made by biopsy in all patients, and we performed this immunophenotyping in all patients before treatment. Because diagnostic accuracy significantly affects the results in studies of such rare diseases, we preferred to conduct this study at a single institution with at least 10 rheumatologists. In addition, because this study was conducted according to the HIP proposed by the National Institute of Allergy and Infectious Disease [24], the power was too weak to detect trends in the immune phenotypes, apart from the conventional ones. In particular, we could not assess the importance of CD4-positive CTLs in the pathogenesis of IgG4-RD, which has been described recently [13]. However, the immune phenotypes that had been assessed differently at each institution were standardized in the HIP, and this facilitated the comparison of reports from different institutions and of immune phenotypes by disease. The most difficult aspect of human immunology is to determine whether the finding is a cause or consequence. Although many suggestive phenomena were observed in the present study, they were mostly correlations, beyond which anything is purely speculation. However, as with many other studies, if the therapeutic target molecules are detected, the phenomena observed in the present study may reveal themselves as either causes or consequences. In conclusion, comprehensive immunophenotyping revealed the importance of plasmablasts and Tfh cells in IgG4-RD. Furthermore, the phenotypes of immune cells in the peripheral blood were found to reflect changes in local lesions. The results suggested that Tfh cells induced the differentiation of B cells into plasmablasts and played a major role in the pathogenesis of IgG4-RD. These findings suggested not only that B cell depletion therapy could be theoretically useful, but also that searching for molecules involved in the interaction between Tfh and B cells might lead to the development of future treatment strategies specifically designed for the pathological features of IgG4-RD. Acknowledgements The author thanks all medical staff at all institutions for providing the data. The author thanks Kahoru Noda for helping with the immunohistochemistry staining and Narumi Sakaguchi for helping with flow cytometric analysis. Funding: This work was supported in part by a Grant-In-Aid for Scientific Research from the Ministry of Health, Labor and Welfare of Japan, the Ministry of Education, Culture, Sports, Science and Technology of Japan, and the University of Occupational and Environmental Health, Japan, through UOEH Grant for Advanced Research. Disclosure statement: S.K. has received speaking fees from Bristol-Myers, Pfizer and Takeda. Y.T. has received consulting fees, speaking fees and/or honoraria from Abbvie, Chugai, Daiichi-Sankyo, Bristol-Myers, Mitsubishi-Tanabe, Astellas, Takeda, Pfizer, Teijin, Asahi-kasei, YL Biologics, Sanofi, Janssen, Eli Lilly and GlaxoSmithKline and has received research grants from Mitsubishi-Tanabe, Takeda, Daiichi-Sankyo, Chugai, Bristol-Myers, MSD, Astellas, Abbvie and Eisai. All other authors have declared no conflicts of interest. Supplementary data Supplementary data are available at Rheumatology online. References 1 Kamisawa T, Zen Y, Pillai S, Stone JH. IgG4-related disease. Lancet  2015; 385: 1460– 71. http://dx.doi.org/10.1016/S0140-6736(14)60720-0 Google Scholar CrossRef Search ADS PubMed  2 Kamisawa T, Shimosegawa T, Okazaki K et al.   Standard steroid treatment for autoimmune pancreatitis. Gut  2009; 58: 1504– 7. http://dx.doi.org/10.1136/gut.2008.172908 Google Scholar CrossRef Search ADS PubMed  3 Umehara H, Okazaki K, Masaki Y et al.   Comprehensive diagnostic criteria for IgG4-related disease (IgG4-RD), 2011. Mod Rheumatol  2012; 22: 21– 30. http://dx.doi.org/10.3109/s10165-011-0571-z Google Scholar CrossRef Search ADS PubMed  4 Deshpande V, Zen Y, Chan JK et al.   Consensus statement on the pathology of IgG4-related disease. Modern Pathol  2012; 25: 1181– 92. http://dx.doi.org/10.1038/modpathol.2012.72 Google Scholar CrossRef Search ADS   5 Ota M, Katsuyama Y, Hamano H et al.   Two critical genes (HLA-DRB1 and ABCF1) in the HLA region are associated with the susceptibility to autoimmune pancreatitis. Immunogenetics  2007; 59: 45– 52. Google Scholar CrossRef Search ADS PubMed  6 Ota M, Umemura T, Kawa S. Immunogenetics of IgG4-related AIP. Curr Top Microbiol Immunol  2017; 401: 35– 44. Google Scholar PubMed  7 Mattoo H, Mahajan VS, Della-Torre E et al.   De novo oligoclonal expansions of circulating plasmablasts in active and relapsing IgG4-related disease. J Allergy Clin Immunol  2014; 134: 679– 87. http://dx.doi.org/10.1016/j.jaci.2014.03.034 Google Scholar CrossRef Search ADS PubMed  8 Wallace ZS, Mattoo H, Carruthers M et al.   Plasmablasts as a biomarker for IgG4-related disease, independent of serum IgG4 concentrations. Ann Rheum Dis  2015; 74: 190– 5. http://dx.doi.org/10.1136/annrheumdis-2014-205233 Google Scholar CrossRef Search ADS PubMed  9 Iwata S, Saito K, Hirata S, Tanaka Y. Phenotypic changes of lymphocyte in a patient with IgG4-related disease after corticosteroid therapy. Ann Rheum Dis  2012; 71: 2058– 9. http://dx.doi.org/10.1136/annrheumdis-2012-201657 Google Scholar CrossRef Search ADS PubMed  10 Achour A, Simon Q, Mohr A et al.   Human regulatory B cells control the TFH cell response. J Allergy Clin Immunol  2017; 140: 215– 22. http://dx.doi.org/10.1016/j.jaci.2016.09.042 Google Scholar CrossRef Search ADS PubMed  11 Lin W, Jin L, Chen H et al.   B cell subsets and dysfunction of regulatory B cells in IgG4-related diseases and primary Sjogren's syndrome: the similarities and differences. Arthritis Res Ther  2014; 16: R118. Google Scholar CrossRef Search ADS PubMed  12 Sumimoto K, Uchida K, Kusuda T et al.   The role of CD19+ CD24high CD38high and CD19+ CD24high CD27+ regulatory B cells in patients with type 1 autoimmune pancreatitis. Pancreatology  2014; 14: 193– 200. Google Scholar CrossRef Search ADS PubMed  13 Mattoo H, Mahajan VS, Maehara T et al.   Clonal expansion of CD4+ cytotoxic T lymphocytes in patients with IgG4-related disease. J Allergy Clin Immunol  2016; 138: 825– 38. Google Scholar CrossRef Search ADS PubMed  14 Maehara T, Mattoo H, Ohta M et al.   Lesional CD4+ IFN-γ+ cytotoxic T lymphocytes in IgG4-related dacryoadenitis and sialoadenitis. Ann Rheum Dis  2017; 76: 377– 85. Google Scholar CrossRef Search ADS PubMed  15 Mattoo H, Della-Torre E, Mahajan VS, Stone JH, Pillai S. Circulating Th2 memory cells in IgG4-related disease are restricted to a defined subset of subjects with atopy. Allergy  2014; 69: 399– 402. Google Scholar CrossRef Search ADS PubMed  16 Della Torre E, Mattoo H, Mahajan VS et al.   Prevalence of atopy, eosinophilia, and IgE elevation in IgG4-related disease. Allergy  2014; 69: 269– 72. http://dx.doi.org/10.1111/all.12320 Google Scholar CrossRef Search ADS PubMed  17 Tanaka A, Moriyama M, Nakashima H et al.   Th2 and regulatory immune reactions contribute to IgG4 production and the initiation of Mikulicz disease. Arthritis Rheum  2012; 64: 254– 63. http://dx.doi.org/10.1002/art.33320 Google Scholar CrossRef Search ADS PubMed  18 Zen Y, Fujii T, Harada K et al.   Th2 and regulatory immune reactions are increased in immunoglobin G4-related sclerosing pancreatitis and cholangitis. Hepatology  2007; 45: 1538– 46. http://dx.doi.org/10.1002/hep.21697 Google Scholar CrossRef Search ADS PubMed  19 Maehara T, Moriyama M, Nakashima H et al.   Interleukin-21 contributes to germinal centre formation and immunoglobulin G4 production in IgG4-related dacryoadenitis and sialoadenitis, so-called Mikulicz's disease. Ann Rheum Dis  2012; 71: 2011– 9. http://dx.doi.org/10.1136/annrheumdis-2012-201477 Google Scholar CrossRef Search ADS PubMed  20 Akiyama M, Yasuoka H, Yamaoka K et al.   Enhanced IgG4 production by follicular helper 2 T cells and the involvement of follicular helper 1 T cells in the pathogenesis of IgG4-related disease. Arthritis Res Ther  2016; 18: 167. http://dx.doi.org/10.1186/s13075-016-1064-4 Google Scholar CrossRef Search ADS PubMed  21 Grados A, Ebbo M, Piperoglou C et al.   T cell polarization toward TH2/TFH2 and TH17/TFH17 in patients with IgG4-related disease. Front Immunol  2017; 8: 235. Google Scholar CrossRef Search ADS PubMed  22 Khosroshahi A, Bloch DB, Deshpande V, Stone JH. Rituximab therapy leads to rapid decline of serum IgG4 levels and prompt clinical improvement in IgG4-related systemic disease. Arthritis Rheum  2010; 62: 1755– 62. Google Scholar CrossRef Search ADS PubMed  23 Carruthers MN, Topazian MD, Khosroshahi A et al.   Rituximab for IgG4-related disease: a prospective, open-label trial. Ann Rheum Dis  2015; 74: 1171– 7. http://dx.doi.org/10.1136/annrheumdis-2014-206605 Google Scholar CrossRef Search ADS PubMed  24 Maecker HT, McCoy JP, Nussenblatt R. Standardizing immunophenotyping for the Human Immunology Project. Nat Rev Immunol  2012; 12: 191– 200. Google Scholar CrossRef Search ADS PubMed  25 Masaki Y, Sugai S, Umehara H. IgG4-related diseases including Mikulicz's disease and sclerosing pancreatitis: diagnostic insights. J Rheumatol  2010; 37: 1380– 5. http://dx.doi.org/10.3899/jrheum.091153 Google Scholar CrossRef Search ADS PubMed  26 Nakayamada S, Kubo S, Yoshikawa M et al.   Differential effects of biological DMARDs on peripheral immune cell phenotypes in patients with rheumatoid arthritis. Rheumatology  2018; 57: 164– 74. Google Scholar CrossRef Search ADS PubMed  27 Kubo S, Nakayamada S, Yoshikawa M et al.   Peripheral immunophenotyping identifies three subgroups based on T cell heterogeneity in lupus patients. Arthritis Rheumatol  2017; 69: 2029– 37. Google Scholar CrossRef Search ADS PubMed  28 Zhao J, Kubo S, Nakayamada S et al.   Association of plasmacytoid dendritic cells with B cell infiltration in minor salivary glands in patients with Sjogren's syndrome. Mod Rheumatol  2016; 26: 1– 9. Google Scholar CrossRef Search ADS PubMed  29 Stone JH, Zen Y, Deshpande V. IgG4-related disease. N Engl J Med  2012; 366: 539– 51. http://dx.doi.org/10.1056/NEJMra1104650 Google Scholar CrossRef Search ADS PubMed  30 Miyake K, Moriyama M, Aizawa K et al.   Peripheral CD4+T cells showing a Th2 phenotype in a patient with Mikulicz's disease associated with lymphadenopathy and pleural effusion. Mod Rheumatol  2008; 18: 86– 90. Google Scholar CrossRef Search ADS PubMed  31 Miyoshi H, Uchida K, Taniguchi T et al.   Circulating naive and CD4+CD25high regulatory T cells in patients with autoimmune pancreatitis. Pancreas  2008; 36: 133– 40. Google Scholar CrossRef Search ADS PubMed  32 Crotty S. Follicular helper CD4 T cells (TFH). Annu Rev Immunol  2011; 29: 621– 63. http://dx.doi.org/10.1146/annurev-immunol-031210-101400 Google Scholar CrossRef Search ADS PubMed  33 Akiyama M, Suzuki K, Yamaoka K et al.   Number of circulating follicular helper 2 T cells correlates with IgG4 and interleukin-4 levels and plasmablast numbers in IgG4-related disease. Arthritis Rheumatol  2015; 67: 2476– 81. http://dx.doi.org/10.1002/art.39209 Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2017. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com

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RheumatologyOxford University Press

Published: Mar 1, 2018

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